【作者】 赵静；

【导师】 董汉松；

【作者基本信息】 南京农业大学 ， 植物病理学， 2004， 硕士

【摘要】 核黄素是人和动植物必需的维生素之一，它的基本生物功能是以单核苷酸（FMN）或黄素腺嘌呤二核苷酸（FAD）的形式作为辅酶，与黄素蛋白质（flavoproteins）或金属黄素蛋白质（metalloflavoproteins）组成复酶，在生物氧化过程中广泛地起着递氢作用，参与动植物、微生物以氧化-还原反应为特征的生理生化过程。核黄素可以诱导植物抗病性，这个过程不需要水杨酸（SA），但需要蛋白质激酶的参与和NPR1／NIM1基因的调节，因此与SA介导的系统获得抗病性（SAR）可能无关，而与蛋白激酶级联关系密切。这些反应是否与核黄素在植物组织内含量的变化有关，还不清楚。 LS基因（Lumizine Synthase）编码二氧四氢蝶啶合酶，该酶催化核黄素生物合成的倒数第二步。LS基因表达程度会影响植物体内核黄素的含量，因此，LS基因是否被不同激发子诱导表达及表达的模式，将能说明核黄素在信号传导中的作用。 作者采用了两种方法来研究核黄素在植物抗病性中的作用，以及核黄素介导的抗病防卫信号通路与上述抗病防卫信号基本通路的关系。第一种方法是研究植物外源施用不同激发子后，LS基因表达与核黄素含量的变化；第二种方法是将LS基因转入拟南芥，研究LS基因在植物体内过量表达对抗病性的影响。 以前的研究表明，核黄素可以诱导拟南芥对Pseudomonas syringae pv．toamato菌株DC3000的抗性，并与多功能激发子harpin_Ea和elicitin诱导的抗病性有许多相似之处。为了研究核黄素信号与抗病防卫其他信号的关系，测定了SA、茉莉酸甲酯（MeJA）、乙烯前体ACC、多功能激发子harpin_Ea和elicitin处理对DC3000在植物中生长的影响。实验结果表明，激发子处理的拟南芥组织内细菌生长量较对照（灭菌水处理的植株）减少，五种外源激发子与核黄素一样，能有效诱导拟南芥对P.syringae的抗性。因此研究它们是否影响核黄素生物合成，可以说明核黄素是否影响基本防卫信号通路。 用RT-PCR技术对五种激发子处理后拟南芥LS基因表达情况进行了检测。LS是组成型表达的，但表达量随激发子处理而发生变化。处理后24h，SA诱导的LS表达量逐步增加；MeJA诱导的LS表达量在12h内基本不变，之后逐渐增加；harpin_Ea诱导后LS的表达量先在6h内下降，之后逐步增加；ACC诱导后LS的表达量先在12h内下降，然后回升到组成型表达的水平；elicitin诱导后LS基因的表达量在6h内增加，后随时间的增加下降。这些结果说明，LS的表达受五种激发子影响，可能与SA、JA、核黄素生物合成中的一个关键基因和植物抗病性关系的研究乙烯信号通路有一定联系. 为了证明LS基因表达与核黄素含量的关系，作者测定了不同激发子处理对核黄素在植物组织内总含量的影响。激发子处理后核黄素的总童都有所变化，不同激发子诱导的情况不同，其中sA和MJ处理以后24h内，核黄素含量逐步增加;ACC和h娜in处理后6h内含量下降，6一24h内含量又逐渐增加;elicitin比较独特，在6h内含量略有增加，之后含量呈下降趋势.说明五种激发子对组织内核黄素产生童的影响有所不同. 其次，我们把LS基因构建到含花椰菜花叶病毒355启动子的转化单元上，然后转化拟南芥.经过一系列筛选，目前仅获得一株再生拟南芥.作者对载体构建、转化和筛选条件进行了细致研究，优化了有关条件。 根据以上研究结果，启动SA、JA、乙烯信号通路的激发子均可刺激LS基因的表达，使核黄素含量提高.同时，它们与核黄素一样，均诱导对细菌的杭性。因此核黄素在组织内含童的变化可能影响植物防卫基本信号通路.显然，本研究的主要缺点有两个.第一，缺少关于LS表达、核黄素含量变化与杭病性之间因果关系的证据，缺少关于核黄素影响基本信号通路的直接证据.一个客观原因是目前还没有核黄素合成受损的植物突变体，同时缺少核黄素合成抑制剂.第二，LS转基因工作所花时间比预计长了一年，因为实验过程中遇到了预料的困难，所以及时把关注点转到优化有关条件的研究。更多还原

【Abstract】 Riboflavin (vitamin 82) is required for microbes, plants, human and other animals.The vitamin plays an important role in plant disease resistance and may regulate plant growth as well. Riboflavin is an activator of a novel signaling pathway leading to systemic resistance , which requires protein kinases and the NIM1/NPR1 protein, but does not require accumulation of SA. On the other hand, lumizine synthase (LS) catalyses the penultimate step of riboflavin biosynthesis. Whether the expression of the gene LS is induced by elicitors that induce disease resistance is unclear. In this study, the author determined two approaches are used to determine contents of riboflavin and LS expression after plants were treated with different elicitors including salicylic acid, jasmonic acid and ethylene, which mediate basal plant defense pathways. In addition, it was planned to determine effects of LS overexpressing on disease resistance in transgenic Arabidopsis plants.Previous studies have shown that treatment of Arabidopsis plants with riboflavin induces resistance to Pseudomonas syringae pv. tomato. The process is similar to that induced by the multiple-functional elicitors harpin and elicitin. The author determined harpin and elicitin, together with SA, methyl jasmonate (MeJA), and the ethylene precursor ACC, for effects on the bacterial growth in plants. Results showed that bacterial population in plants treated with these materials was markedly lower than that in control plants treated with water. Therefore, determining effects of these elicitors on riboflavin biosynthesis could indicate whether riboflavin affects basal defense pathways.Effects of the five elicitors on LS gene expression was investigated. Although the gene had a low level of constitutive expression, based on assays of control plants, its expression pattern and extents were affected differently by treatment with elicitors. Treatment with SA caused an increase in LS transcripts in 24 hours. MeJA treatment increased the gene expression after 12 hours of treatment. The gene transcription increased in 6 hours after the application of harpinEa- However, treatment with ACC orelicitin decreased levels of gene expression in 6 and 12 hours, respectively. Thereafter, LS gene expressed in plants treated with ACC or elicitin similarly as did in control plants.The author determined whether treatment with the five elicitors affects production of free riboflavin in plants consistently with expression of the LSgene. Riboflavin increased levels in plants treated SA or MeJA after 24 hours of treatment. In contrast, treatment with elicitin caused decrease in riboflavin concentration after 6 hours. Interestingly, treatment with ACC or harphiEa decreased riboflavin levels in 6 hours but an evident increase thereafter. Clearly, changes of endogenous riboflavin levels are consistent with changes of the LS gene expression in plants treated with different elicitors.Finally, the LS gene was cloned the transformation vector containing the 35S promoter, and introduced into Arabidopsis plants. A LS -transgenic Arabidopsis line had been generated; more lines are being screened. Transgenic plants that overexpress the LS gene will be investigated to determine whether genetic modulation of riboflavin biosynthesis affects pathogen defense and relevant processes.In conclusion, treatment of Arabidopsis plants with SA, JA and ethylene stimulates the LS gene expression. This process is coincident with increase in riboflavin levels in the plants. Therefore modulation of endogenous riboflavin levels is associated with the basal defense pathways mediated differentially by SA, JA and ethylene.更多还原